The virus of Yellow Fever (YF) is considered the prototype of the Flaviviridae family, also represented by several other medically important viruses that cause serious diseases such as Dengue, Japanese Encephalitis and West Nile Fever (Barrett, 2002). According to World Health Organization (WHO) more than 200,000 cases of YF infection, including 30,000 deaths occur annually worldwide (90% of disseminated cases in Africa). The safest strategy for disease prevention remains vaccination, whereas there is still no effective drug against infection by YF. Over the past 70 years more than 400 million people globally were vaccinated with YF-attenuated virus (17DD), considered very safe and effective. Despite the success of mass vaccination with 17DD, which is capable of inducing both lasting response of neutralizing antibody as cytotoxic T cell response (Poland, Calisher et al., 1981; Reinhardt, Jaspert et al., 1998), adverse severe events (as a result of vaccination) has been systematically reported in the literature [reviewed in (Liu, 2003)]. In some cases, immunization has been directly associated with increased severity of symptoms (Monath, Arroyo et al., 2002) and may even lead to fatal reactions (Vasconcelos, Luna et al., 2001; Lefeuvre, Marianneau et al., 2004). In this scenario the development of new vaccination strategy, such as DNA vaccines encoding specific viral sequences (Donnelly, Ulmer et al., 1997; Lewis and Babiuk, 1999; Robinson, 1999; Schultz, Pavlovic et al., 2000) is of fundamental importance for the development of even safer vaccine strategies.
The genome of Yellow Fever Virus (YFV) is arranged in a positive mRNA molecule, approximately 10.8 Kb flanked by the structures of 5 ‘cap and 3rd handle’ terminal not poly adenilada. The RNA YFV encodes three structural genes (Capsid—C, Membrane—M and Envelope—E) and 7 genes that encode non-structural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b and NS5). During assembly of virus the carboxy-terminal domain of protein C acts as a signal sequence for translocation of the precursor PreM/M into the lumen of the endoplasmic reticulum (ER) of the host cell, allowing proper maturation of the M and E protein. Co-expression of proteins PreM/M and flavivirus, in mammalian cells results in the formation of pseudo-viral particles capable of inducing humoral response (Raviprakash, Kochel et al., 2000; Wu, Li et al. 2006), because the protein E is the major target for neutralizing antibodies.
Co-expression of proteins PreM/M and E, as vaccine strategy, has been described as being capable of inducing the neutralizing antibodies productions against the virus of Japanese Encephalitis and Dengue (Konishi, Yamaoka et al., 1998; Konishi, Yamaoka et al., 2000; Konishi, Ajiro. et al, 2003). However, these vaccines failed to induce long-term response with appropriate titers of neutralizing antibodies (Lu, et al Raviprakash, 2003). The inefficiency of these formulations is probably related to the mechanism of presentation of these antigens to the immune system of hosts. Most of endogenously produced antigens, characteristic of DNA vaccines, are kidnapped and presented to the host immune system by MHC I molecules. For a satisfactory immune response, with high neutralizing antibodies production, it is critical that the antigens are presented to cells of the T helper CD4+ type by MHC II molecules. The processing and the antigen presentation by MHC II induces the activation of T CD4+ cells that is vital to the functioning of genetic vaccines, as has already been demonstrated in studies of deletion of MHC II (Raviprakash, Marques et al., 2001) and CD4+ depletion in mice (Lu et al Raviprakash, 2003). The activation of CD4+ cells is essential for the induction of CD8+ response, development of memory cells (Marques, Chikhlikar et al., 2003) and clonal expansion of antigen-specific B cell (De Arruda, Chikhlikar et al., 2004). So that antigens endogenously produced are directed to molecules of class II, instead class I, it is necessary that these proteins are fused to peptides signs which direct them for the lysosomal compartment of the cell.
The possibility of directing endogenously produced antigens, for processing via MHC II was strongly increased after the discovery of a type I transmembrane protein, called Lysosome-Associated Membrane Protein—LAMP (Chen, Murphy et al. 1985). LAMP is a protein that binds to the outer membrane of the lysosome via its carboxy-terminal sequence YXXØ, present in a cytoplasmic tail of 11 amino acids (Guarnieri, Arterburn et al., 1993; Rohrer, Schweizer et al., 1996; Obermüller, Kiecke et al., 2002). The LAMP intracellular traffic includes specialized multilaminar compartments of immature Antigen-Presenting Cells (APC), called MIIC and CIIV, where processing and formation of antigenic peptide-MHC II complex takes place (Kleijmeer, Morkowski et al. 1997; Drake, Lewis et al., 1999; Turley, Inaba et al., 2000). The finding of colocalization of LAMP and MHC II molecules allows its use as support for chimeric antigen, containing the sequences of LAMP targets, aimed at direction antigen processing for MHC II compartment. Many works have demonstrated that antigens fused to the LAMP (antigen/LAMP) are capable of generating a higher proliferative activity of specific antigen lymphocytes, high titers of antibodies and intense cytotoxic T activity in relation to the wild non-fused antigens to the LAMP (Rowell, Ruff et al., 1995; Wu, Guarneri et al., 1995; Ruff, Guarneri et al., 1997; Raviprakash, Marques et al., 2001; Su, Vieweg et al., 2002; Donnelly, Berry et al., 2003; Anwar, Chandrasekaran et al., 2005).
The attenuated virus vaccine 17DD has been produced on the campus of Manguinhos—FIOCRUZ/RJ since 1937, i.e. at least 70 years. The mass immunization with the vaccine 17DD, as well as the systematic fight against the transmission vector of Yellow Fever (Aedes aegypti), were and remains crucial strategies for disease control in the country. Despite the efficacy and safety of the vaccine 17DD, it is not recommended for infants, pregnant women, and people who have immunodeficiencies and who are allergic to egg proteins (substrate for vaccine 17DD production). It is estimated that approximately 5% of the population presents allergies and/or side effects in response to the vaccine, possibly culminating in rare cases of death caused by vaccination.
Recently, facing the death of monkeys in wildlife regions where YFV circulates, the population began to panic at the speculations of the re-introduction of urban Yellow Fever in the country. Considering the risk of infection prevalent in tropical areas, the invasion of the urban environment by the vector of the disease, global warming and the lack of appropriate policies to combat the vector insect, the risk of spread of the disease in urban areas cannot be neglected. The chaos caused by dengue in the state of Rio de Janeiro, which by the way is transmitted by the same vector of Yellow Fever, illustrates the risk of a possible (but not likely) outbreak of urban Yellow Fever in the country. Considering all these factors, the development of a complementary vaccine strategy, and/or alternative against Yellow Fever, can complement/replace the use of attenuated virus vaccine version.
Although no DNA vaccine has been approved for human use, this type of technology has been increasingly enhanced and potentially shall replace the formulations based on living microorganisms. DNA-based formulations can be easily handled and dosed, require no special temperature condition for storage and distribution and even eliminate any possible risk of infection by the live/attenuated agents. This type of technology also allows the handling of immunogens able to stimulate the immune system with specific epitopes and biological signals, avoiding the use of unnecessary and potentially harmful antigens/epitopes regarding to possible cross-immune responses (main obstacle to the development of an effective vaccine against dengue, due to cross-reaction between its 4 serotypes). Finally, with the technological advancements of tools for manipulation of microorganisms and purification of molecules on a large scale, DNA vaccines might be produced on a larger scale and with a lower final cost when compared to the attenuated/inactivated formulations.
It is important to note that before the encouraging significantly results obtained by our group, using a genetic vaccine based on the viral sequence of the E protein fused to the LAMP, and improving this vaccine by the optimization of antigens for the expression in humans, we believe in the possibility of developing and implementation of a DNA vaccine, even more secure, able to confer immunity against the virus of Yellow Fever in humans. This type of technology might also serve as subsidy to the development of other viral vaccines, especially against other flaviviruses such as Dengue virus.